1. Field of the Invention
The present invention relates to a method for preparing a solid support surface having binding agents, such as analyte-specific ligands, immobilized thereto, and more particularly to such a surface which resists non-specific binding. The invention also relates to the use of the prepared solid support surface in analysis, such as immunogenicity assays, and to a protein-resistant solid support surface for coupling of binding agents thereto.
2. Description of the Related Art
A variety of analytical techniques are used to characterize interactions between molecules, particularly in the context of assays directed to the detection and interaction of biomolecules. For example, antibody-antigen interactions are of fundamental importance in many fields, including biology, immunology and pharmacology. In this context, many analytical techniques involve binding of a “ligand”, such as an antibody, to a solid support, followed by contacting the ligand with an “analyte”, such as an antigen. Following contact of the ligand and analyte, some characteristic is measured which is indicative of the interaction, such as the ability of the ligand to bind the analyte. It is often desired that after measurement of the interaction, it should be possible to dissociate the ligand-analyte pair in order to “regenerate” free ligand, thereby enabling reuse of the ligand surface for a further analytical measurement.
Analytical sensor systems that can monitor such molecular interactions in real time are gaining increasing interest. These systems are often based on optical biosensors and usually referred to as interaction analysis sensors or biospecific interaction analysis sensors. A representative such biosensor system is the BIACORE® instrumentation sold by Biacore AB (Uppsala, Sweden), which uses surface plasmon resonance (SPR) for detecting interactions between molecules in a sample and molecular structures immobilized on a sensing surface. With the BIACORE® systems it is possible to determine in real time without the use of labeling not only the presence and concentration of a particular molecule in a sample, but also additional interaction parameters such as, for instance, the association rate and dissociation rate constants for the molecular interaction.
Since the SPR-based detection, like several other so-called label-free detection techniques, senses mass changes at the sensor surface, non-specific binding to the sensor surface will also be sensed, giving a false addition to the detected response at the surface. This is particularly the case where the samples are complex mixtures such as a blood serum or crude cell extract. The non-specific binding may arise from binding of non-analyte molecules in the sample to the immobilized ligand or from non-specific binding of analyte or non-analyte molecules to the actual sensor surface. In, for example, immunogenicity studies where antibodies in serum samples are analyzed, problems in most cases arise due to non-specific binding from non-analyte species in the serum, this non-specific binding often being greater than the specific binding of the target antibody.
It is known that poly(ethylene glycol) (PEG) coatings may significantly reduce the non-specific adsorption of proteins and cells to a surface.
U.S. Pat. No. 6,475,808 discloses an assay device comprising a substrate with a surface having an array of discrete array-regions. An ordered hydrophobic monolayer of alkyl chains is chemisorbed or physisorbed to the surface, and a hydrophilic monolayer formed from poly(ethylene glycol) chains is covalently linked to the hydrophobic monolayer. A plurality of protein-immobilizing groups are covalently attached to a selected fraction of the poly(ethylene glycol) chains within the array regions. The hydrophobic monolayer and the poly(ethylene glycol) chains are effective in combination to resist non-specific protein binding.
WO 2004/005477 discloses a microarray which comprises a substrate having a substantially planar surface comprising an organic-chemically modified dielectric-coated reflective metal, e.g., gold, and a plurality of proteins stably attached to the surface via a chemical adapter, e.g., a functionalized dextran. After spotting the proteins onto the substrate surface, the surface may be derivatized with poly(ethylene glycol) or a poly(ethylene glycol) analogue to inhibit non-specific protein adsorption.
WO 03/005890 discloses an optical fiber surface plasmon resonance (SPR) sensor having a gold surface to which a dextran layer is bound via a self-assembled monolayer (SAM) of 11-mercapto-dodecanol. Anti-myoglobin antibodies are attached to the dextran via carboxylated hydroxyl groups thereof. To eliminate non-specific binding to the sensor, thiol-terminated poly(ethylene glycol), e.g., methoxy-PEG-thiol, may be coupled to the gold surface through a gold-thiol bond or to the dextran. Immobilized PEG surrounding the sensor will prevent non-specific interactions with the surface while allowing specific receptor-ligand interactions.
The present invention seeks to provide an improved method for preparing a protein-resistant solid support surface having binding agents immobilized thereon.